1. Field of the Invention
The present invention relates to measuring and detecting coagulation and coagulation-related activities in fluids, particularly human blood. More particularly, the present invention relates to an improved activated clotting time test for whole blood which provides accurate and reliable detection of coagulation-related activities regardless of the concentration of heparin in the blood. Further, this invention relates to a new and improved technique for evaluating the effects of therapeutic levels of platelet function inhibitors on the coagulation of whole blood regardless of the heparin concentration of the blood.
2. Description of the State of the Art
Blood coagulation is a complex chemical and physical reaction which occurs when blood comes into contact with an activating agent, such as an activating surface or an activating reagent. In accordance with one simplified conceptual view, the whole blood coagulation process can be generally viewed as three activities: agglutination of platelets, blood clotting, and fibrous tissue formation. In vivo, platelets flow through the blood vessels in an inactivated state because the blood vessel lining, the endothelium, prevents activation of platelets. When a blood vessel is damaged, however, the endothelium loses its inert character and platelets are activated by contact with tissue underlying the damaged site. Activation of the platelets causes them to become "sticky" and adhere together. Additional platelets then adhere to the activated platelets and also become activated. This process continues until a platelet "plug" is formed. This platelet plug then serves as a matrix upon which blood clotting proceeds.
If the chemical balance of the blood is suitable, thrombin is then produced which causes conversion of fibrinogen to fibrin, which forms the major portion of the clot mass. During clotting, additional platelets are activated and trapped in the forming clot, contributing to clot formation. As clotting proceeds, polymerization and cross-linking of fibrin serves as the permanent clot. Thus, platelet activation plays a very important function in blood coagulation.
A number of different medical apparatuses and testing methods exist for measuring and determining coagulation and coagulation-related activities of blood. These apparatuses and methods provide valuable medical information to an attending physician. For example, the information assists a physician in prescribing medication, predicting post-operative bleeding and prescribing various therapies. Some of the more successful techniques of evaluating blood clotting and coagulation are the plunger techniques illustrated by U.S. Pat. Nos. 4,599,219 to Cooper et al., 4,752,449 to Jackson et al., and 5,174,961 to Smith, all of which are assigned to the assignee of the present invention, and all of which are incorporated herein by reference.
Automated apparatuses employing the plunger technique for measuring and detecting coagulation and coagulation-related activities generally comprise a plunger sensor cartridge or cartridges and a microprocessor controlled apparatus into which the cartridge is inserted. The apparatus acts upon the cartridge and the blood sample placed therein to induce and detect the coagulation-related event. The cartridge includes a plurality of test cells, each of which is defined by a tube-like member having an upper reaction chamber where a plunger assembly is located and where the analytical test is carried out, and a reagent chamber which contains a reagent or reagents. For an activated clotting time (ACT) test, for example, the reagents include an activation reagent to activate coagulation of the blood. A plug member seals the bottom of a reagent chamber. When the test commences, the contents of the reagent chamber are forced into the reaction chamber to be mixed with the sample of fluid, usually human blood or its components. An actuator, which is a part of the apparatus, lifts the plunger assembly and lowers it, thereby reciprocating the plunger assembly through the pool of fluid in the reaction chamber. The plunger assembly descends on the actuator by the force of gravity, resisted by a property of the fluid in the reaction chamber, such as its viscosity. When the property of the sample changes in a predetermined manner as a result of the onset or occurrence of a coagulation-related activity, the descent rate of the plunger assembly therethrough is changed. Upon a sufficient change in the descent rate, the coagulation-related activity is detected and indicated by the apparatus.
Certain discoveries have recently been made which contribute to a better understanding of the role of platelets in an ACT test. Such discoveries suggest that the activation of the platelets has a significant and previously unappreciated effect on ACT test results. While it has long been suspected that platelet activation contributes to total blood coagulation times, until fairly recently, there has been no technique available for confirming and quantifying the impact of platelet activation on ACT. U.S. Pat. No. 5,312,826 to Baugh describes an improved ACT test which includes a platelet activation phase to accommodate the effects of platelet activation. In the platelet activation phase an activating reagent is mixed with a sample of blood to be tested, then the mixture is gently agitated in such a manner and for a period of time sufficient to establish a predetermined and predictable contribution to the ACT from platelet activation. To evaluate platelet function, two simultaneous ACT tests (with different platelet activation phases) are performed, and the difference between the ACTs is indicative of the platelet functionality of the sample of blood. In a further improvement, described in U.S. Ser. No. 08/640,275, filed Apr. 30, 1996, the sample of blood is mixed with a chemical platelet activating agent to facilitate the participation of active platelets in the blood clotting reaction, thereby shortening the clotting time of the blood. If the platelets are inactive or not functioning normally, the activator will have minimal or no effect on the clotting time. Both U.S. Pat. No. 5,312,826 and U.S. Ser. No. 08/640,275 are assigned to the assignee of the present invention, and are incorporated by reference in their entireties herein.
Although previous apparatuses using the plunger sensing technique have proven generally satisfactory, the need for certain enhancements has been identified. Specifically, while these techniques can measure and detect coagulation and coagulation-related activities in a sample of blood, none are designed to conveniently accommodate either heparinized or unheparinized (patient) blood in a single test cartridge. Most of the apparatuses currently available for monitoring platelet function are designed specifically for highly heparinized blood, or the test cartridge used to perform the ACT test must include a sufficient concentration of heparin to inhibit post-platelet activation (i.e., subsequent to factor XII activation) reactions. Heparin is necessary to inhibit reactions which occur later in the coagulation process, such as activation of factors X, II (prothrombin) and I (fibrinogen), i.e., reactions in the common pathway or in a later stage in the intrinsic pathway. By inhibiting these later clotting factors, heparin (which together with antithrombin III accelerates the inactivation of factor X and thrombin) helps to accentuate the contribution of platelets to clotting, which is the rate-limiting step in blood coagulation, i.e., the actual clotting times in the ACT test depend on how rapidly the platelets are able to activate clotting. Accordingly, heparin must be either present in the blood sample or included in the test cartridge to inhibit non-platelet-related reactions which tend to obscure the rate-limiting activation step.
Although heparin is effective in accentuating the platelet activation step, the concentration of heparin varies widely between blood samples, and thus is a variable which must be accounted for in coagulation-based platelet function assays. Because heparin is highly effective in preventing and treating thrombosis and pulmonary embolism, it is commonly administered to patients as an anticoagulant in a variety of circumstances and in a wide range of medical dosages. For example, in a cardiovascular surgical situation such as a pulmonary bypass surgical operation, relatively high therapeutic dosages of anticoagulant (typically between 3.5 and 7.0 units of heparin per milliliter of blood) are administered to prevent the blood from clotting in the extracorporeal bypass circuit and in the patient's body as a result of environmental changes brought on by the surgery. The concentration of heparin in the patient's blood at any given time during this surgical operation depends on the stage of the procedure, with generally high levels present during the procedure and typically no or negligible levels prior to and following surgery. Low-dose heparin therapy, on the other hand, is used in a wide variety of clinical applications, where the clotting time of the blood must be slightly extended and confined within a relatively narrow range. Low-dose heparin treatment usually involves administering between 0.1 and 1.0 units of heparin per milliliter of blood.
Thus, to accommodate these various circumstances and heparin dosages, commercial coagulation-based platelet function assays must include a variety of test cartridge types, with the various cartridges comprising different amounts of heparin. Special test cartridges must also be produced to accommodate blood samples which contain excessive amounts of heparin, since the clotting times for these samples will frequently exceed the test period for the ACT-based analysis. To reduce the clotting time of highly heparinized blood samples, an additional reagent such as protamine is included in the test cartridge to neutralize a portion of the heparin. In addition to the extra cost associated with these multiple cartridge types, hospital and laboratory personnel must maintain a complete inventory of test cartridges, and then select the correct cartridge type for each blood sample to be analyzed. The selection and retrieval of test cartridges can be particularly inconvenient in surgical and clinical procedures which require sequential diagnostic tests, for example, during cardiopulmonary bypass surgery. For example, coagulation-based platelet function measurements are typically taken before the administration of heparin for cardiopulmonary bypass surgery to establish the patient's baseline platelet function and clotting time, and periodically throughout surgery to ensure adequate heparinization and to monitor platelet function. The ACT values can also be used to monitor the neutralization of heparin by protamine where a return to preoperative ACT is typically used as the target value. A different cartridge type is currently required to measure platelet function at these different stages of the surgical procedure.
Another deficiency with existing techniques is their inability to monitor the effectiveness of therapeutic levels of antiplatelet reagents or platelet function inhibitors in a variety of circumstances. While tests exist which can assist physicians in evaluating the efficacy of drugs and pharmacological agents at inhibiting the normal functions of platelets, none can accommodate either heparinized or unheparinized blood in a single test cartridge. In addition, interactions between heparin and antiplatelet reagents can interfere with the calculated platelet function.
A need therefore exists for an improved coagulation-based platelet function test, and particularly one based upon an improved activated clotting time test, which can accurately and reliably detect coagulation and coagulation-related activities regardless of the heparin content of the blood. The ability to measure and evaluate platelet activation and function independent of heparin concentration is important, particularly during invasive cardiac procedures in which the anticoagulant content of the blood varies significantly throughout the procedure. At the present time, there is no known single apparatus or method which is universally applicable to both heparinized and unheparinized blood. A need also exists for an improved activated clotting time test which can monitor the effectiveness of therapeutic levels of platelet function inhibitors in a variety of circumstances, regardless of the heparin content of the blood. Until this invention, no such devices or methods existed.